Approximately 80% of hepatocellular carcinoma, a leading cause of cancer-related deaths, is caused by infection with either hepatitis B or hepatitis C virus (HCV). The burden of HCV infection worldwide is very large, with most of the personal and economic burden yet to come, as cirrhosis and cancer take years to develop. Approximately 170 million people are infected worldwide with HCV. Most infected individuals do not clear the disease, but develop chronic infections that often lead to end-stage liver disease. Current treatment is limited to co-treatment with ribavirin and interferon 1, a therapy that is expensive and ineffective in 50% of infected individuals. Therefore, there is an urgent need to identify new and accessible viral and cellular targets for therapies against HCV. This application will study two important HCV RNA-protein interactions that regulate HCV gene amplification. A highly-structured RNA element is located in the viral 5' noncoding region that functions as an internal ribosome entry site (IRES) that can directly recruit 40S ribosomal subunits to the viral genome. Specifically, we will employ biochemical, NMR and single-molecule fluorescence approaches to explore the timing and control of subunit joining on IRES-initiated mRNAs. In addition, we will examine roles for eukaryotic initiation factors eIF5B and eIF2 in causing conformational changes in the ribosomal subunits. Using FRET and optical tweezer approaches, we will measure stabilities of IRES-ribosomal complexes and rearrangements of these complexes on mRNAs. Proposed long-range communication between 5' and 3' ends in HCV viral RNAs will be studied using NMR. Secondly, we will study the interaction of RNA helicase RIG-I with viral and host RNA ligands. Specifically, a cell-based crosslinking-immunoprecipitation (CLIP) assay will be employed to identify HCV and cellular RNA ligands for RIG-I. Using an infectious HCV cell-based system, we will determine whether RIG-I targets the viral RNA in replicating or translating RNA molecules. Using siRNA-mediated gene knock down, biochemical and cell-biological assays, we will characterize roles, 5' end modifications and localization of identified RIG-I ligands. Using biochemical assays, NMR and FRET analyses, biophysical and structural parameters for RIG-I ligand recognition and enzymatic kinetics that are activated by the RNAs will be examined and a structural framework for these activities will be established. Structural, dynamic and mechanistic views of HCV non-coding region structures, that modulate translation, and that of specific RNA ligands associated with RIG-I, that are part of innate immune responses, will be merged with approaches that probe function, and potential inhibition by therapeutics. PUBLIC HEALTH RELEVANCE: An estimated 170 million people worldwide and 4 million in the United States are infected with hepatitis C virus (HCV). The majority of patients do not resolve the infection and become chronic carriers, ultimately needing expensive liver transplants. There is no vaccine for HCV, and current treatments, which include ribavirin and interferon 1, are expensive and relatively ineffective. It was discovered that HCV binds ribosomes by a novel, unprecedented mechanism of internal ribosome binding, and that a key defense protein against viruses, helicase RIG-I, interacts with the viral genome. This proposal explores whether the mechanisms by which ribosome and RIG-I bind to the viral RNA genome present an Achilles heel that can be used for antiviral intervention. To do this, novel biochemical, structural and genetic approaches will be employed to study protein-HCV RNA interactions.